Affiliation

Abstract

In metazoans, how replication origins are specified and subsequently activated is not well understood. Drosophila amplicons in follicle cells (DAFCs) are genomic regions that undergo rereplication to increase DNA copy number. We identified all DAFCs by comparative genomic hybridization, uncovering two new amplicons in addition to four known previously. The complete identification of all DAFCs enabled us to investigate these in vivo replicons with respect to parameters of transcription, localization of the origin recognition complex (ORC), and histone acetylation, yielding important insights into gene amplification as a metazoan replication model. Significantly, ORC is bound across domains spanning 10 or more kilobases at the DAFC rather than at a specific site. Additionally, ORC is bound at many regions that do not undergo amplification, and, in contrast to cell culture, these regions do not correlate with high gene expression. As a developmental strategy, gene amplification is not the predominant means of achieving high expression levels, even in cells capable of amplification. Intriguingly, we found that, in some strains, a new amplicon, DAFC-22B, does not amplify, a consequence of distant repression of ORC binding and origin activation. This repression is alleviated when a fragment containing the origin is placed in different genomic contexts.

Figures

Figure 1.

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Genome-wide analysis of Drosophila follicle…

Figure 1.

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Genome-wide analysis of Drosophila follicle cell amplicons by aCGH identifies two new amplified…

Figure 1.

Genome-wide analysis of Drosophila follicle cell amplicons by aCGH identifies two new amplified regions. DNA from 16C follicle cells was competitively hybridized with diploid embryonic DNA to microarrays with approximately one probe every 600 bp by aCGH. The log2 ratio of follicle cell DNA compared with embryonic DNA is shown on the Y-axis. Entire chromosome arms are shown in A. The newly identified amplicons DAFC-22B and DAFC-34B are marked in boxes. (B) A 150-kb view of DAFC-22B. CG7337 spans the entire length of the amplified region. (C) A 150-kb view of DAFC-34B. Vm34Ca encodes a structural component of the vitelline membrane. The gap spaces reflect repetitive DNA regions or nonunique sequences in the Drosophila reference genome sequence that are not included on the microarray.

Figure 2.

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Gene expression and localization of…

Figure 2.

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Gene expression and localization of RNAPII, ORC, and tetra-acetylated H4 at the six…

Figure 2.

Gene expression and localization of RNAPII, ORC, and tetra-acetylated H4 at the six amplicons. (A–F) Two-hundred-kilobase regions of all follicle cell amplicons with sequence reads from RNA-seq of purified follicle cells, as well as localization of RNAPII, ORC, and tetra-acetylated H4 by ChIP–chip from stage 10 egg chambers. Maximum sequence read is 100 for DAFC-22B, DAFC-30B, and DAFC-62D, whereas maximum sequence read is 1000 for DAFC-34B, DAFC-66D, and DAFC-7F. ChIP–chip experiments depict the log2 ratios of immunoprecipitated DNA compared with input DNA, which contains increased DNA copy levels at the amplicons. (E,F) The chorion amplicons show the greatest increase in DNA copy number, indicating the most rounds of origin firing, which correspond to the largest zones of ORC enrichment and overlap of these regions with tetra-acetylated H4 enrichment. Bars below the ORC and tetra-acetylated H4 values represent significant regions of enrichment, as calculated by the MA2C program, with a P-value cutoff of 0.002 and a bandwidth of 5000 bp for ORC, and P-value cutoff of 0.00001 and a bandwidth of 500 bp for tetra-acetylated H4.

Figure 3.

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Gene amplification is not required…

Figure 3.

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Gene amplification is not required for high follicle cell gene expression. ( A…

Figure 3.

Gene amplification is not required for high follicle cell gene expression. (A) Gene expression by RNA-seq was analyzed in 100-kb windows, and the 90th percentile of highly expressed 100-kb regions was plotted and is designated by black boxes. Chromosome 3L is depicted in A. Many genomic regions show high expression but are not amplified. (B,C) Two-hundred-kilobase regions of two nonamplified genomic loci with highly expressed genes. Sequence reads from RNA-seq of purified follicle cells as well as localization of RNAPII, ORC, and tetra-acetylated H4 by ChIP–chip from stage 10 egg chambers are shown as in Figure 2. 7C contains the defective chorion 1 gene (dec-1), and 26A contains a cluster of genes encoding components of the vitelline membrane. Bars below the tetra-acetylated H4 values represent significant regions of enrichment, as calculated by the MA2C program, using a P-value cutoff of 0.00001 and a bandwidth of 500 bp.

Figure 4.

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ORC localizes to genomic regions…

Figure 4.

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ORC localizes to genomic regions that do not become amplified. ( A ,…

Figure 4.

ORC localizes to genomic regions that do not become amplified. (A,B) Two-hundred-kilobase regions of two nonamplified, ORC-bound regions. Sequence reads from RNA-seq of purified follicle cells as well as localization of ORC and tetra-acetylated H4 by ChIP–chip from stage 10 egg chambers are shown as in Figure 2. ORC localizes to the transcription unit of white in genomic region 3B and mei-S332 in 58B, although neither gene is highly expressed (RPKM, <5). (C) Features of ORC-bound regions showing the fraction of the 99 ORC-bound regions that overlap with amplified domains, transcription units, and expressed genes (RPKM, >3).

Figure 5.

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Tethering Rpd3 to the amplification…

Figure 5.

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Tethering Rpd3 to the amplification reporter TT1 eliminates its amplification and reduces H4K8…

Figure 5.

Tethering Rpd3 to the amplification reporter TT1 eliminates its amplification and reduces H4K8 acetylation. (A) qPCR with transgene-specific primers was used to determine copy number of the TT1 transgene in different genetic backgrounds and experimental conditions. TT1 amplification is repressed upon Rpd3 expression (heat shock) and tethering to TT1 at stage 10 and pooled stages 11 and 12 egg chamber samples. (B) Levels of H4K8 acetylation at TT1 and representative primers from endogenous amplicons in stage 10 by ChIP-qPCR. The arrow points to reduction in acetylated H4K8 levels upon Rpd3 expression and tethering to TT1.

Figure 6.

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DAFC-22B exhibits strain-specific amplification that…

Figure 6.

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DAFC-22B exhibits strain-specific amplification that correlates with the ability of the region to…

Figure 6.

DAFC-22B exhibits strain-specific amplification that correlates with the ability of the region to bind ORC. (A,B) Follicle cell DNA copy levels as well as ORC and tetra-acetylated H4 by ChIP-chip from stage 10 egg chambers are shown as in Figure 2 for two different strains: OrRTOW and OrRMOD. DAFC-22B is not amplified in the OrRMOD strain, and this region does not show ORC localization. Tetra-acetylated H4 is observed in similar patterns in both amplifying and nonamplifying strains. (C) cDNA was generated from different egg chamber stages and analyzed by qPCR. Fold enrichment is relative to Rps17 and normalized to the stage 1–9 sample. There is no difference in expression levels for the gene in DAFC-22B (CG7337) for any developmental stage.

Genetic analysis of cis control elements for differential DAFC-22B amplification reveals distant repression of origin activation. (A) The DAFC-22B regions for P-element-mediated transformation and testing sufficiency for amplification are shown in black bars. Fragment A was PCR-amplified from OrRTOW (amplifying) and OrRMOD (nonamplifying) flies. Fragment B was amplified from OrRTOW flies. These sequences integrated into random sites in the genome of a strain that does not amplify the endogenous 22B locus. ORC2 ChIP–chip data are shown for OrRTOW. ORC2 ChIP-seq data from KC cells are also shown (scale is sequence tag density). The circle represents the position of qPCR primers that detect amplification of fragment A. The star represents the position of qPCR primers that detect amplification of fragment A or B. (B) The TOW A fragment shows amplification using both sets of primers. The stage 1–8 egg chambers serve as a control, because amplification does not begin until stage 10. (C) The TOW B fragment shows amplification using only the star set of primers because the endogenous 22B sequence recognized by the circle primers does not amplify. (D) The MOD A fragment shows amplification using both sets of primers. (E) ORC2 ChIP-qPCR was performed on stage 10 egg chambers from transgenic flies carrying the MOD A fragment. The primers designated 22B endogenous do not amplify the MOD A transgene.